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Abstract

Background

We reported that the compositions of arachidonic acid (ARA) in erythrocytes and plasma
phospholipids (PL) in the elderly were lower than those in the young, though the ARA
intake was nearly identical.

Objective

We further analyzed data in four study groups with different ages and sexes, and determined
that the blood ARA levels were affected by the kinds of dietary fatty acids ingested.

Methods

One hundred and four healthy young and elderly volunteers were recruited. Dietary
records together with photographic records from 28 consecutive days were reviewed
and the fatty acid composition in plasma lipid fractions and erythrocyte PL was analyzed.

Results

No correlations for ARA between dietary fatty acids and blood lipid fractions were
observed. A significant negative correlation between eicosapentaenoic acid (EPA) +
docosahexaenoic acid (DHA) intake and ARA composition in erythrocyte PL was observed.
ARA composition in erythrocyte PL was significantly lower in elderly subjects than
in young subjects, because EPA and DHA intake in elderly subjects was higher than
in young subjects. However, after removing the effect of dietary EPA+DHA intake, the
ARA composition in erythrocyte PL in elderly subjects was significantly lower than
that in young subjects.

Conclusions

Changes in physical conditions with aging influenced the low ARA composition of erythrocyte
in elderly subjects in addition to the effects of dietary EPA and DHA.

Background

Arachidonic acid (ARA), one of the n-6 polyunsaturated fatty acids (PUFA), is the
predominant fatty acid (FA) of membrane phospholipids (PL) in mammalian brain and
neural tissues [1,2]. ARA rapidly accumulates in the human brain during the growth spurt that starts at
the beginning of the third trimester of pregnancy and remains in high demand until
about 2 years of age [3,4].

Many studies in the last decade have shown the role of sufficient intake of n-3 PUFA
in the prevention of several diseases, particularly coronary heart disease [5-7]. Eicosanoids made from ARA are generally more potent mediators of inflammation, vasoconstriction,
and platelet aggregation than those made from the eicosapentaenoic acid (EPA) of n-3
PUFA, so a lower intake of vegetable oil containing abundant linoleic acid (LA), the
precursor of ARA, has been recommended. However, a recent epidemiological study has
indicated that the levels of ARA in blood do not coincide precisely with the incidence
of inflammatory diseases [8,9]. The risk of colorectal cancer was inversely associated with erythrocyte compositions
of docosahexaenoic acid (DHA), ARA, and PUFA among Japanese men and women [10]. The current study found a U-shaped relationship between blood cell ARA content and
acute coronary syndrome case status, which means that the odds for this disease tended
to be lower in the second and third quartiles as compared with the first and highest
quartiles [11].

Hereafter, we think that our attention will be focused on blood ARA for the associations
with disorders; therefore, we should understand how the blood ARA is changed by factors
including the discrepancies in diet, sex, and age. Weseler et al. [12] reported that dietary supplementation with 200 mg ARA increased ARA in erythrocyte
membrane PL in nursing mothers. Our previous data showed that the intake of a capsule
containing only 80 mg ARA, which is roughly equivalent to 60% of the usual daily ARA
intake, increased the ARA level in all lipid fractions of the blood [13]. Thus, the blood ARA level is markedly increased by dietary intervention with ARA.
However, in the studies targeted at a free-living population, previous reports have
not coincided in the correlations with dietary ARA and blood ARA composition: these
data have not necessarily shown a positive correlation and several data sets have
shown a negative correlation or no correlation at all [14-18].

We conducted a dietary survey using dietary records together with photographic records
over 28 consecutive days and determined the FA compositions in plasma triacylglycerol
(TG), esterified cholesterol (EC), and PL, and erythrocyte membrane PL in four study
groups: young men, young women, elderly men, and elderly women. We reported that the
compositions of ARA in plasma and erythrocyte PL in the elderly were lower than those
in the young, though the ARA intake was nearly identical [19]. In this study, we further analyzed data in four study groups with different ages
and sexes, and determined whether the blood ARA levels were affected by the kinds
of dietary FA ingested.

Subjects and methods

Subjects

The details of the subjects are reported in our previous study [13]. Briefly, groups of healthy subjects in this study were as follows: young men group
(YM-G), young women group (YW-G), elderly men group (EM-G), and elderly women group
(EW-G). The YM-G consisted of 20 men who were 20 years of age. The YW-G consisted
of 30 women who were 20 years of age. The EM-G consisted of 22 men who were 60-75
years of age. The EW-G consisted of 32 women who were 56-73 years of age. The subjects
in the four study groups received a verbal explanation of the study and provided written
informed consent prior to participating in the study. The study was performed after
obtaining approval from the medical ethics review board of Kagawa Nutrition University.

Dietary assessment

A dietary record was continuously maintained using a written form together with photographic
records for 28 consecutive days. Although every investigation was conducted in early
summer, the investigation year was 2004 for the young women, 2005 for the elderly
men, 2007 for the elderly women, and 2008 for the young men. The details of the dietary
survey method are reported in our previous study [20]. Weights of food consumed were estimated from the daily dietary records and food
images. The Fifth Revised and Enlarged Edition of the Tables of Fatty Acid Composition
[21] in Japan was used as a reference for FA intake.

Fatty acid analysis of blood lipid fractions

Fasting blood sampling was conducted on the day after completion of the 28-day dietary
survey. After blood sampling, the samples were centrifuged to separate plasma and
erythrocytes. The preparation of erythrocyte membranes and the analysis of FA compositions
were conducted as described previously [13]. Briefly, erythrocytes were washed with saline, and after Tris-HCl buffer was added,
they were centrifuged to obtain the erythrocyte membranes. The total FA of the erythrocyte
membranes and plasma were extracted, the FA of plasma TG, EC, and PL and erythrocyte
membrane PL were separated by thin-layer chromatography and, after transmethylation,
the FA composition was analyzed by gas chromatography. The FA compositions were calculated
as percentages of the total FA.

Statistical analysis

The relationships between dietary FA and FA in plasma TG, EC, and PL and erythrocyte
membrane PL were examined by Spearman's rank correlation. Statistical differences
among the four groups were determined using ANOVA and the Bonferroni post hoc test.
We calculated the least square means and 95% confidence intervals of ARA composition
in erythrocyte PL using analysis of covariance (ANCOVA) models, with EPA+DHA intakes
as the covariables and ARA composition in erythrocyte PL as the dependent variable,
because it was possible that dietary EPA+DHA intake affected the ARA composition in
erythrocyte PL.

A significant difference in analysis results was observed at P < 0.05. We conducted calculations using the Statistical Package for Social Science
software (SPSS for Windows, version 17.0; Chicago, IL, USA).

Results

Spearman's correlation coefficients between dietary EPA, DHA and ARA intake and the
corresponding FA composition of plasma TG, EC, and PL and erythrocyte PL in YM-G,
YW-G, EM-G, EW-G, and the total subjects group (ALL-G) are shown Table 1. Significant positive correlations between dietary intake and all blood lipid fractions
were observed for EPA and DHA in ALL-G. No correlations for ARA were observed between
dietary ARA and all blood lipid fractions of YM-G, YW-G, EM-G, and EW-G.

A scatter plot between dietary EPA+DHA intake and ARA compositions in erythrocyte
PL is shown in Figure 1. For YM, YW, EM, and EW, EPA+DHA intake and ARA composition in erythrocyte PL, significant
negative correlations were observed. Regression lines between men and women overlapped
in both the young and elderly groups, while regression lines between young and elderly
were parallel in the men and women groups.

Because dietary EPA+DHA intake seems to affect the ARA composition in erythrocyte
PL, we analyzed by ANCOVA with EPA+DHA intakes as the covariables. As a result, the
F value of common regression was 40.8 (P < 0.001), the slope of the regression line was -2.3, and the F value of non-parallelism
was 0.54 (P = 0.65). As a result, the effect of EPA+DHA intake on ARA compositions in erythrocyte
PL was not negligible and the regression lines in YM, YW, EM and EW were parallel.
We then calculated the adjusted mean and standard error of the mean (SEM) of ARA composition
in erythrocyte PL in YM-G, YW-G, EM-G, and EW-G; these results are shown in Table
3. Adjusted ARA compositions in erythrocyte PL were not significantly different between
men and women in both young and elderly subjects; those in the elderly men and women
were significantly lower compared with those in the young men and women (P < 0.001).

Table 3. The mean and SEM of EPA+DHA and ARA intakes and ARA composition in erythrocyte PL
among YM-G, YW-G, EM-G, and EW-G

Discussion

We determined the relationships between dietary FA estimated from dietary records
together with photographic records over 28 consecutive days and the compositions of
ARA in plasma TG, EC, and PL and erythrocyte PL in young men and women and elderly
men and women. Our present study showed that: (1) dietary ARA intakes were not correlated
with the composition of ARA in erythrocyte PL, but dietary EPA and/or DHA intakes
were negatively correlated with the composition of ARA in erythrocyte PL in all subjects
groups and (2) after removing the effect of dietary EPA+DHA intake, the ARA composition
in erythrocyte PL was significantly lower in elderly subjects than in young subjects.

In previous survey among Japanese people, a negative correlation between ARA intake
and serum PL level of ARA was observed [14]. Conversely, Kuriki et al. [15] reported that ARA in young and elderly groups demonstrated positive correlations
between dietary compositions (wt%) and plasma compositions (wt%). Other studies indicated
that the intake of ARA was not significantly related to ARA level in the plasma PL
[16,17]. Similarly, Sun et al. [18] showed no correlation between the compositions of ARA in plasma and erythrocytes
and ARA intake measured with the food-frequency questionnaire in 306 US women. Thus,
in the studies targeted at free-living populations, previous reports did not agree
on the results of the correlations with dietary ARA and blood ARA composition. In
this study, we clearly demonstrated that dietary EPA and/or DHA intakes, but not dietary
ARA intakes, markedly affected the ARA compositions in blood ARA levels. This may
be one of the reasons that assessment of the ARA level in blood from dietary FA is
difficult.

Yanagisawa et al. [22] reported an assessment of the serum and erythrocyte FA compositions in groups of
Japanese people stratified by age and they indicated that ARA levels of blood lipid
fractions in elderly people were lower compared with those in young people. In 530
Yup'ik Eskimos who were 14 to 94 years old, elderly subjects consumed more traditional
foods than younger subjects did and in those who consumed traditional foods, the EPA
and DHA compositions of red blood cell membranes were significantly higher and ARA
composition was significantly lower [23]. Our data also indicated that the compositions of EPA and DHA of plasma and erythrocyte
PL in the elderly were significantly higher than those in the young, and those of
ARA were significantly lower [19]. We indicated the possibility of the displacement and inhibition of incorporation
of ARA by dietary EPA and DHA in blood PL in elderly subjects. In this paper, the
negative correlations between EPA+DHA intake and the ARA composition of erythrocyte
PL in all subject groups were observed, so the above hypothesis was more strongly
supported by our analysis. In addition, we conducted the analysis of ANCOVA, with
EPA+DHA intake as the covariable. After removing the effect of dietary EPA+DHA intake,
the adjusted ARA composition in erythrocyte PL was significantly lower in elderly
subjects than in young subjects. Therefore, in addition to the effects of dietary
EPA and DHA, we estimated that changes in physical conditions with aging affected
the low ARA composition of erythrocyte in elderly subjects.

Erythrocyte membrane FA composition is affected by diet and is considered to reach
a new steady state level 4 to 5 weeks after the establishment of new diet management
[24]. We performed a dietary investigation for 28 consecutive days using dietary records
together with photographic records to precisely assess habitual FA intakes in order
to determine the association between dietary FA and ARA composition in erythrocyte
PL. Different conditions, such as a shorter period than our dietary survey period
or different dietary survey methods, may not bring about the desired results of the
relationship between dietary and erythrocyte ARA. Our present results were obtained
under a precise dietary survey for 1 month.

In summary, the ARA levels in blood all lipid fractions, especially in erythrocyte
PL, were affected by the amount of EPA and/or DHA intakes. The ARA composition in
erythrocyte PL was significantly lower in elderly subjects than in young subjects,
because EPA and DHA intakes in elderly subjects were higher than in young subjects.
However, after removing the effect of dietary EPA+DHA intake, the adjusted ARA composition
of erythrocyte PL in elderly subjects was significantly lower than that in young subjects.
Consequently, changes in physical conditions with aging influenced the low ARA composition
of erythrocyte in elderly subjects in addition to the effects of dietary EPA and DHA.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TK conceived the study, participated in the design of the study, acquired data, performed
the statistical analysis and drafted the manuscript. SH, TH, NA, YK, NI and KK carried
out the survey, measured blood fatty acid compositions, and organized the data. EA,
HK and YK participated in the design of the study and helped conducting the study.
All authors read and approved the final manuscript.

References

Sinclair AJ, Crawford MA: The accumulation of arachidonate and docosahexaenoate in the developing rat brain.